Field of the Invention
The present invention is broadly concerned with cryopreserved self-sustaining bodies formed from compositions of cells, methods of forming the same, and methods of using the same to produced pooled preparations of cells.
Description of Related Art
Hepatocytes are parenchymal liver cells, and make up 60-80% of the cytoplasmic mass of the liver. Hepatocytes play a key role in the detoxification, modification, and excretion of exogenous and endogenous substances. One of the detoxifying functions of hepatocytes is to modify ammonia to urea for excretion. They are also important in protein synthesis and storage, in the transformation of carbohydrates, and in the synthesis of cholesterol, bile salts, and phospholipids.
Viable intact hepatocytes, isolated from human or laboratory animal livers, offer an experimental model for phase I and phase II drug metabolism studies, as well as enzyme induction studies. Isolated and cultured hepatocytes are also an appropriate model for studying overall liver function. Fresh hepatocytes are obtainable only from liver resections or non-transplantable livers of organ donors. Thus, the availability of viable, fresh liver tissue from humans is inconsistent and overall fairly limited, thus limiting the ability to conduct experiments using such a system, because availability does not always coincide with when such cells are needed. When tissue does become available, the isolated hepatocytes must be cryopreserved and banked for later use. However, individual hepatocyte samples have limited applicability due to individual variation in cell function. For example, due to individual variability in Cytochrome P450 (CYP) expression, studying enzyme induction using hepatocytes isolated from a single donor is usually not representative of a given population's response to a particular new chemical entity (NCE) or drug. Thus, hepatocyte preparations pooled from multiple donors are desirable for studying NCE's, as such pooled preparations provide a composite or “average” hepatocyte preparation. It is therefore desirable to accumulate a bank of cryopreserved hepatocytes from various donors for pooling. In a traditional method of pooling hepatocytes, selected frozen vials from the individual donor bank are thawed, pooled together, and then refrozen. Depending upon the desired pool, all or only a portion of the thawed aliquot is used in the pool. This frozen pooled product is then used for NCE studies.
Cryopreservation is a process where cells or whole tissues are preserved by cooling to low sub-zero temperatures (at least about −90° C.). At these low temperatures, any biological activity, including the biochemical reactions that would lead to cell death, is effectively stopped. Several methods have been used to successfully cryopreserve hepatocytes obtained from laboratory animals and humans. Methods of cryopreservation vary, but cryopreservation of hepatocytes in a medium containing 20-90% fetal bovine serum (FBS) and 10-20% dimethyl sulfoxide (DMSO) as a cryoprotectant yield a high viable cell recovery upon thawing. Density of the hepatocytes in the freezing media may vary from 106 to 107 cells/mL. While the development of cryopreservation methods for the storage of hepatocytes has significantly facilitated the availability of human hepatocytes, cryopreservation has been found to cause significant decrease in cellular viability after thawing. Controlled slow rate freezing minimizes the formation of intracellular ice-crystals, which play a large role in hepatocyte damage during the cryopreservation procedure. Rapid thawing of frozen hepatocytes at 37° C. has also been shown to improve viable cell recoveries. However, the poor recovery of cells following cryopreservation and thawing continues to limit the use of hepatocytes for in vitro liver models. This problem is particularly apparent in traditional pooled hepatocyte preparations, which are prepared using multiple freeze-thaw cycles, where each successive freeze-thaw cycle causes increased damage to at least a portion of the hepatocytes in the preparation, reducing overall cell viability of the resulting pool. Similar problems are encountered during cryopreservation and storage of other cellular and subcellular fractions, such as organelles.
Accordingly, the need remains for processes that would enable the availability of hepatocytes and other organelles for medical research, clinical testing, induction studies, and other purposes. A need further exists for pooled hepatocytes and cellular and subcellular fraction having stable and reproducible characteristics and acceptable viability. In addition, there is a need for pooled products that avoid cryoinjury caused by multiple freeze-thaw cycles on the properties and characteristics of the cells.